FIGURE 7 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

Dabrafenib increases oxidative metabolism in MDSCs. A, Heat map of the average log₂FC in gene expression for the genes indicated ± 1.5 µmol/L DAB clusters identified in MDSC scRNA sequencing data described in Fig 6. Positive values indicate genes with induced expression by DAB, while negative values show genes that are downregulated by DAB. For representation, log₂FC = 0 indicates clusters that were excluded from the analysis with FindAllMarkers function (gene expressed in <10% cells and/or log₂FC ±0.25). Statistics show Wilcoxon test. * = adjusted P-value <0.05. B, Representative flow cytometry histogram of CellROX Green (ROS in mitochondria and nucleus) in wild-type MDSC generated with 1.5 µmol/L DAB (red line) or control (black line). Panels show geometric mean fluorescence intensity (gMFI) ±SD (n = 4). P value was determined by two-tailed unpaired Student t test. Significance considered P < 0.05. Experiment was repeated three times with similar results. C, qPCR of genes related to oxidative respiration (Cox6a2), fatty acid metabolism (Pparg), and glycolysis (Aldh18a1, Slc2a1, Slc16a3, Ldha) in total wild-type and GCN2−/− MDSC lysates, normalized to 18s mRNA expression(n = 4). P values were determined by two-way ANOVA with Šidák correction post-test. Significance considered P < 0.05. Experiment was repeated three times with similar results. D, Quantification of oxidative respiration by OCR and glycolysis by ECAR, by Seahorse assay. Lines represent the mean ±SEM of four independent experiments. Gluc = Glucose; OM = Oligomycin; FCCP = Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot/AA = Rotenone/Antimycin. E, Maximum respiration calculated from OCR data and maximum glycolytic capacity calculated from ECAR data. Bars represent mean ± SEM of four independent experiments. P values were determined by two-way ANOVA with Šidák correction post-test. Significance considered P < 0.05. DAB = dabrafenib.